Interferon (IFN) and IFN-stimulated gene products (ISGs) are essential components of the innate immune response to viral infection. Conversely, viruses need to limit the induction or function of ISGs for successful infection of their respective host. One of the most highly prevalent human pathogens is the beta-herpesvirus human cytomegalovirus (HCMV), yet it is not clear how HCMV activates and modulates the IFN response. The goal of this application is therefore to identify and characterize cellular activators and viral modulators of this innate immune response to HCMV. We have shown that the activation of interferon-regulatory factor 3 (IRF3) is essential for HCMV-induced IFN and ISG induction. However, we also observed that virion proteins of the non-human primate virus rhesus CMV (RhCMV) prevent IRF3 activation by HCMV. Two proteins of the RhCMV tegument, pp65a and pp71 inhibit IRF3-dependent ISG induction. Unexpectedly, pp71 (UL82) of HCMV also inhibited IRF3-dependent ISG transcription. Interestingly, human fibroblasts stably transfected with pp72 (UL82-HF) were completely refractory to IRF3 activation by HCMV, but not by Vesicular Stomatitis Virus or poly-IC. In addition, double stranded, interferon-stimulatory DNA (ISD) failed to activate IRF3 in UL82-HF. ISD-dependent IRF3 activation is a recently described novel innate immune response pathway that is independent of toll-like receptors or the dsRNA-sensors RIG-I and MDA-5. Therefore, we hypothesize that HCMV and ISD share a common IRF3-activating signal transduction pathway that is modulated by CMV tegument proteins. To test this hypothesis we plan to identify host cell factors required for HCMV and/or ISD- dependent IRF3 activation. This will be achieved in a high-throughput screen of small intefering RNAs targeting approximately 17,000 known and unknown genes. We will further identify the mechanism of viral IRF3-inhibtion by identifying the step within the signal transduction cascade that is targeted by tegument proteins of RhCMV and HCMV. Finally, we will characterize the role of viral tegument proteins in modulating IRF3 activation during viral infection. Ultimately, these in vitro results will allow us to test the importance of modulating the innate immune response in an emerging animal model for HCMV.